Jacinta Serpa

Human MUC2 Mucin Gene Is Transcriptionally Regulated by Cdx Homeodomain Proteins in Gastrointestinal Carcinoma Cell Lines

In intestinal metaplasia and 30% of gastric carcinomas, MUC2 intestinal mucin and the intestine-specific transcription factors Cdx-1 and Cdx-2 are aberrantly expressed. The involvement of Cdx-1 and Cdx-2 in the intestinal development and their role in transcription of several intestinal genes support the hypothesis that Cdx-1 and/or Cdx-2 play important roles in the aberrant intestinal differentiation program of intestinal metaplasia and gastric carcinoma. To clarify the mechanisms of transcriptional regulation of the MUC2 mucin gene in gastric cells, pGL3 deletion constructs covering 2.6 kb of the human MUC2 promoter were used in transient transfection assays, enabling us to identify a relevant region for MUC2 transcription in all gastric cell lines. To evaluate the role of Cdx-1 and Cdx-2 in MUC2 transcription we performed co-transfection experiments with expression vectors encoding Cdx-1 and Cdx-2. In two of the four gastric carcinoma cell lines and in all colon carcinoma cell lines we observed transactivation of the MUC2 promoter by Cdx-2. Using gel shift assays we identified two Cdx-2 binding sites at –177/–171 and –191/–187. Only simultaneous mutation of the two sites resulted in inhibition of Cdx-2-mediated transactivation of MUC2 promoter, implying that both Cdx-2 sites are active. Finally, stable expression of Cdx-2 in a gastric cell line initially not expressing Cdx-2, led to induction of MUC2 expression. In conclusion, this work demonstrates that Cdx-2 activates the expression of MUC2 mucin gene in gastric cells, inducing an intestinal transdifferentiation phenotype that parallels what is observed both in intestinal metaplasia and some gastric carcinomas.

Hypercholesterolemia promotes bone marrow cell mobilization by perturbing the SDF-1:CXCR4 axis.

Hypercholesterolemia is associated with elevated peripheral blood leukocytes and increased platelet levels, generally attributed to cholesterol-induced proinflammatory cytokines. Bone marrow (BM) cell mobilization and platelet production is achieved by disrupting the SDF-1:CXCR4 axis, namely with granulocyte colony-stimulating factor and/or CXCR4 antagonists. Here we show that high cholesterol disrupts the BM SDF-1:CXCR4 axis; promotes the mobilization of B cells, neutrophils, and progenitor cells (HPCs); and creates thrombocytosis. Hypercholesterolemia was achieved after a 30-day high-cholesterol feeding trial, resulting in elevated low-density lipoprotein (LDL) cholesterol levels and inversion of the LDL to high-density lipoprotein cholesterol ratio. Hypercholesterolemic mice displayed lymphocytosis, increased neutrophils, HPCs, and thrombocytosis with a lineage-specific decrease in the BM. Histologic analysis revealed that megakaryocyte numbers remained unaltered but, in high-cholesterol mice, they formed large clusters in contact with BM vessels. In vitro, LDL induced stromal cell-derived factor-1 (SDF-1) production, suggesting that megakaryocyte delocalization resulted from an altered SDF-1 gradient. LDL also stimulated B cells and HPC migration toward SDF-1, which was blocked by scavenger receptor class B type I (cholesterol receptor) inhibition. Accordingly, hypercholesterolemic mice had increased peripheral blood SDF-1 levels, increased platelets, CXCR4-positive B lymphocytes, neutrophils, and HPCs. High cholesterol interferes with the BM SDF-1:CXCR4 axis, resulting in lymphocytosis, thrombocytosis, and HPC mobilization.

Infection by Helicobacter pylori expressing the BabA adhesin is influenced by the secretor phenotype

Helicobacter pylori (Hp) infects half the worlds population and causes diverse gastric lesions, from gastritis to gastric cancer. Our aim was to evaluate the significance of secretor and Lewis status in infection and in vitro adherence by Hp expressing BabA adhesin. We enrolled 304 Hp‐infected individuals from Northern Portugal. Gastric biopsies, blood and saliva were collected. Polymerase chain reaction (PCR) and immunofluorescence were used to detect BabA+ Hp in gastric biopsies. In vitro adherence by a BabA expressing Hp strain to gastric biopsies was performed. Secretor status was identified by Ulex, a lectin that recognizes secretor‐dependent glycan structures in saliva and in gastric mucosa, and by Lewisa/b antibodies, and indirectly by identification of an inactivating mutation in the FUT2 gene (G428A). BabA status of infecting Hp was associated with CagA and VacAs1 (p < 0.05), intercellular localization of Hp (p < 0.01) and the presence of intestinal metaplasia (p < 0.05) and degenerative alterations (p < 0.005) in the biopsies. BabA was associated (p < 0.05) with Ulex staining of gastric biopsies and, although not significantly, to absence of homozygosity for FUT2 G428A inactivating polymorphism. In vitro Hp adherence was higher in cases wild‐type or heterozygous for FUT2 G428A mutation (p < 0.0001), cases staining for Ulex (p < 0.0001) and a−b+ and a−b− secretor phenotypes (p < 0.001). In conclusion, BabA+ Hp infection/adhesion is secretor‐dependent and associated with the severity of gastric lesions. Copyright

Mucins as key molecules for the classification of intestinal metaplasia of the stomach

Abstract. Mucins and mucin-associated carbohydrates have a distinct expression pattern that can be modified under pathological conditions. Normal gastric mucosa expresses MUC1 and MUC5AC in foveolar epithelium and MUC6 in the glands. Lewis type-1 chain antigens (Lea and Leb) are expressed in foveolar epithelium, whereas Lewis type-2 chain antigens (Lex and Ley) are expressed in the glands. In this study we used monoclonal antibodies to evaluate the pattern of mucins and Lewis type-1 carbohydrates in intestinal metaplasia (IM) and compared it with IM types determined using histochemistry. In type-I or complete IM we found expression of MUC2 intestinal mucin and decreased/absent expression of MUC1, MUC5AC and MUC6. In type-II/III or incomplete IM there was co-expression of MUC2 and the mucins expressed in the stomach. No major differences were detected among the three IM types regarding expression of Lewis antigens. Furthermore we observed that sialylated compounds other than sialyl-Lea are responsible for histochemical detection of sialomucins and that sulpho-Lea/c is expressed in the presence or absence of sulphomucins detected using histochemistry. We conclude that mucin immunohistochemistry may replace classic histochemistry for the classification of IM into complete and incomplete types. The present study challenges the distinction of type-II from type-III IM since we did not observe major differences in the expression profile of mucins and Lewis type-1 carbohydrates. Finally, it seems necessary to evaluate the predictive value of IM according to the presence of specific sulphated carbohydrates (e.g. sulpho-Lea/c) rather than histochemically detected sulphomucins.

Butyrate-rich Colonic Microenvironment Is a Relevant Selection Factor for Metabolically Adapted Tumor Cells

The short chain fatty acid (SCFA) buyrate is a product of colonic fermentation of dietary fibers. It is the main source of energy for normal colonocytes, but cannot be metabolized by most tumor cells. Butyrate also functions as a histone deacetylase (HDAC) inhibitor to control cell proliferation and apoptosis. In consequence, butyrate and its derived drugs are used in cancer therapy. Here we show that aggressive tumor cells that retain the capacity of metabolizing butyrate are positively selected in their microenvironment. In the mouse xenograft model, butyrate-preselected human colon cancer cells gave rise to subcutaneous tumors that grew faster and were more angiogenic than those derived from untreated cells. Similarly, butyrate-preselected cells demonstrated a significant increase in rates of homing to the lung after intravenous injection. Our data showed that butyrate regulates the expression of VEGF and its receptor KDR at the transcriptional level potentially through FoxM1, resulting in the generation of a functional VEGF:KDR autocrine growth loop. Cells selected by chronic exposure to butyrate express higher levels of MMP2, MMP9, α2 and α3 integrins, and lower levels of E-cadherin, a marker for epithelial to mesenchymal transition. The orthotopic model of colon cancer showed that cells preselected by butyrate are able to colonize the animals locally and at distant organs, whereas control cells can only generate a local tumor in the cecum. Together our data shows that a butyrate-rich microenvironment may select for tumor cells that are able to metabolize butyrate, which are also phenotypically more aggressive.

Two new FUT2 (fucosyltransferase 2 gene) missense polymorphisms, 739G→A and 839T→C, are partly responsible for non-secretor status in a Caucasian population from Northern Portugal

Secretor status is defined by the expression of H type 1 antigen on gastric surface epithelium and external secretions. The H type 1 structure, and other fucosylated carbohydrates (Le(a), sialyl-Le(a), Le(b), Le(x), sialyl-Le(x) and Le(y)), can serve as ligands for several pathogens, including Helicobacter pylori, and are cancer-associated antigens. Secretor individuals are more susceptible to some bacterial and viral infections of the genito-urinary and digestive tracts. The aim of the present study was to examine FUT2 (fucosyltransferase 2 gene) polymorphisms in a Caucasian population of non-secretor individuals (n=36) from northern Portugal and to evaluate the activity of the mutant FUT2 enzymes. The secretor status was determined by UEAI [Ulex europaeus (gorse) lectin] histochemistry in gastric mucosa, and FUT2 polymorphisms were studied by restriction-fragment-length polymorphism and direct sequencing. The majority of non-secretors (88.9%) were homozygous for 428G-->A polymorphism; 5.6% were homozygous for 571C-->T and 5.6% were homozygous for two new missense polymorphisms, 739G-->A (2.8%) and 839T-->C (2.8%). By kinetic studies it was demonstrated that the two new FUT2 mutants (739G-->A and 839T-->C) are almost inactive and are responsible for some non-secretor cases.

Lewis antigen expression in gastric mucosa of children: Relationship with Helicobacter pylori infection

Objective Lewis epithelial antigen expression has a role in Helicobacter pylori adherence, presumably mainly in cag A-positive strains. The authors investigated whether Lewis antigen expression in childrens gastric mucosa was associated with H. pylori infection, cag A status, patient age, or presence of duodenal ulcer (DU). Methods The expression of Lewis A (Lea), B (Leb), X (Lex), and Y (Ley) was detected by immunohistochemistry in the antral and oxyntic mucosae of 70 children. Children were divided in four age groups (<4 years; 4–8 years; 9–12 years; and 13–18 years). Results Forty-seven of the 70 children had H. pylori and 17 had DU. The cag A status was determined by polymerase chain reaction in 34 patients. Lea and Leb were expressed in 64% and 44% of the patients, respectively; Lex and Ley were expressed in the glands in all of the patients and in the superficial epithelium. Leb expression was more common among patients without H. pylori (15/23, 65%) than in those with H. pylori (16/47, 34%) (P = 0.03). In noninfected patients, Leb and superficial Ley expression were associated with increased age. Leb expression was more common in patients with chronic gastritis than in those with DU. Lex superficial expression was significantly associated with DU in patients with H. pylori. Conclusion In children, the expression of Leb and Ley in the superficial gastric epithelium depends on age. Other receptors, such as Lex, may have a role in H. pylori colonization, especially in patients with DU. Studies assessing the expression of Lewis antigens in children may contribute to an understanding of the mechanisms of acquisition of H. pylori infection.

Lewis enzyme (α1–3/4 fucosyltransferase) polymorphisms do not explain the Lewis phenotype in the gastric mucosa of a Portuguese population

AbstractThe human α-1,3/4 fucosyltransferase III (FucT III) catalyses the synthesis of Lewis antigens including Leb antigen which is a ligand for Helicobacter pylori adhesion. Several polymorphisms have been described in the FUT3 gene affecting both the transmembrane and catalytic domains, some of which affect the enzyme activity. The aim of the present work was to study the Lewis gene polymorphisms in a Caucasian Portuguese population, with a high rate of H. pylori infection, and to evaluate the implications of mutant enzymes in Leb expression in the gastric mucosa. We studied 460 asymptomatic or dyspeptic individuals from northern Portugal. Screening for Lewis gene polymorphisms was performed by SSCP and direct sequencing. Lewis phenotype in gastric mucosa was determined by immunohistochemistry. In 47 individuals with a Lewis negative blood group, we found FUT3 gene polymorphisms that were previously described in other populations: 59T>G, 202T>C, 314C>T, 508G>A and 1067T>A. Among the 47 Lewis negative individuals in blood, only nine were also negative in gastric mucosa, suggesting the existence of another α 1-4 fucosyltransferase that is responsible for Lea and Leb synthesis in gastric mucosa.

HNF1β drives glutathione (GSH) synthesis underlying intrinsic carboplatin resistance of ovarian clear cell carcinoma (OCCC).

Chemoresistance to platinum-based antineoplastic agents is a consistent feature among ovarian carcinomas; however, whereas high-grade serous carcinoma (OSC) acquires resistance during chemotherapy, ovarian clear cell carcinoma (OCCC) is intrinsically resistant. The main objective of this study was to explore, in vitro and in vivo, if hepatocyte nuclear factor 1β (HNF1β) and glutaminolysis contribute for the resistance of OCCC to carboplatin through the intrinsically increased GSH bioavailability. To disclose the role of HNF1β, experiments were also performed in an OSC cell line, which does not express HNF1β. Metabolic profiles, GSH quantification, HNF1β, and γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) expression, cell cycle, and death were assessed in ES2 cell line (OCCC) and OVCAR3 cell line (OSC); HNF1β knockdown was performed in ES2 and murine model of subcutaneous and peritoneal OCCC tumors was established to test buthionine sulphoxamine (BSO), as a sensitizer to carboplatin. Glutaminolysis is activated in ES2 and OVCAR3, though ES2 exclusively synthesizes amino acids and GSH. ES2 cells are more resistant to carboplatin than OVCAR3 and the abrogation of GSH production by BSO sensitizes ES2 to carboplatin. HNF1β regulates the expression of GCLC, but not GCLM, and consequently GSH production in ES2. In vivo, BSO prior to carboplatin reduces dramatically subcutaneous tumor size and GSH levels, as well as peritoneal dissemination. Our study discloses HNF1β as the mediator of intrinsic OCCC chemoresistance and sheds a light to re-explore a cancer adjuvant therapeutic approach using BSO to overcome the lack of efficient therapy in OCCC.

STAT3:FOXM1 and MCT1 drive uterine cervix carcinoma fitness to a lactate-rich microenvironment

Uterine cervix cancer is the second most common malignancy in women worldwide with human papillomavirus (HPV) as the etiologic factor. The two main histological variants, squamous cell carcinomas (SCC) and adenocarcinomas (AC), resemble the cell morphology of exocervix and endocervix, respectively. Cancer metabolism is a cancer hallmark conditioned by the microenvironment. As uterine cervix homeostasis is dependent on lactate, we hypothesized lactate plays a role in uterine cervix cancer progression. Using in vitro (SiHa-SCC and HeLa-AC) and BALB-c/SCID models, we demonstrated that lactate metabolism is linked to histological types, with SCC predominantly consuming and AC producing lactate. MCT1 is a key factor, allowing lactate consumption and being regulated in vitro by lactate through the FOXM1:STAT3 pathway. In vivo models showed that SCC (SiHa) expresses MCT1 and is dependent on lactate to grow, whereas AC (HeLa) expresses MCT1 and MCT4, with higher growth capacities. Immunohistochemical analysis of tissue microarrays (TMA) from human cervical tumors showed that MCT1 expression associates with the SCC type and metastatic behavior of AC, whereas MCT4 expression concomitantly increases from in situ SCC to invasive SCC and is significantly associated with the AC type. Consistently, FOXM1 expression is statistically associated with MCT1 positivity in SCC, whereas the expression of FOXO3a, a FOXM1 functional antagonist, is linked to MCT1 negativity in AC. Our study reinforces the role of the microenvironment in the metabolic adaptation of cancer cells, showing that cells that retain metabolic features of their normal counterparts are positively selected by the organ’s microenvironment and will survive. In particular, MCT1 was shown to be a key element in uterine cervix cancer development; however, further studies are needed to validate MCT1 as a suitable therapeutic target in uterine cervix cancer.